Rapid developments in genomics, proteomics, and combinatorial chemistry have reshaped the field of drug discovery, providing new drug targets for selective screens and new compounds to be tested in those screens. While combinatorial methods have given rise to large libraries of compounds, typically these compounds result in improved lead candidates that must undergo further transformations by conventional medicinal chemistry to yield new drug candidates. Bioengineering, in the context of high-throughput combinatorial methodologies, has not impacted lead optimization nearly as much as it has lead discovery, mainly because of the highly selective, intricate chemistries often required to optimize lead compounds and the lack of a suitably broad high-throughput platform. Combinatorial biocatalysis can help overcome these obstacles by exploiting the exquisite selectivity and unique reactivity of enzymes and microbial biocatalysts; however, to date this technology has been limited to the derivatization of soluble substrates. We propose to expand the scope of combinatorial biocatalysis to include reactions on, and the generation of libraries from, lead molecules attached to solid and soluble polymer supports. In the process, we will develop a high-throughput, biocatalytic technology for drug discovery. The specific aims are: 1. To expand the breadth of biocatalysis on solid- and polymer-supported compounds in aqueous and nonaqueous media; 2. To develop strategies for attaching lead compounds and removing their derivatives from solid and polymeric supports; 3. To demonstrate high-throughput, combinatorial biocatalytic lead optimization of complex natural and synthetic molecules, screen resulting derivatives for biological activity, and scale up structurally and functionally interesting derivatives using biotransformations. A series of lead molecules will be used in this work, ranging from enzyme substrates that are attached onto solid and soluble polymer supports to complex compounds (the flavonoid bergenin and the current HIV-1 protease inhibitor indinavir). Successful completion of this research program will result in a powerful methodology that can be used by biomedical investigators in the search for new, more potent small molecule therapeutics.